Establishment And Application Of Chromosomal Copy Number Variations Detection Based On High-throughput Sequencing

BackgroundCri du Chat Syndrome and Angelman syndrome is caused by chromosomal microdeletion and microduplication(belonging to chromosomal copy number variation),accounting for birth defects of 1-1.7%.And the spontaneous miscarriage rate due to abnormal chromosomes was above 50%,and chromosomal copy number variation accounted for 10%.At present,the main diagnostic ways is the gold standard karyotyping analysis,but the drawback is the low resolution,invasive,time-consuming procedure.High-resolution microarray comparative genomic hybridization(array-CGH)is considered the gold standard of to detect CNVs below 5Mb,but with the drawbacks as demanding sample quality,invasive and expensive.High-throughput sequencing technology has been used in clinical laboratory noninvasive prenatal detection of chromosomal aneuploidy,the technology of low cost,high resolution,less demanding on the sample and the potential of testing chromosomal microdeletion and microduplication for invasive prenatal diagnosis.The aim of this study was to establish a high-throughput sequencing platform using low-depth sequencing to detect CNV and evaluate the consistency between sequencing and array-CGH in identification of CNV in miscarriages and mental retardation.And its application in the simulated maternal plasma samples detected common diseases such as Cri du Chat Syndrome and other common birth defects.Method:1.Obtained 5 Mb reads of each sample,quality control,cancel duplication reads,aligned to reference genomes,portioned 50kb for each window along the genome.Then Circular Binary Segmentation(CBS)algorithm and Hidden Markov Model were adopted to determine breakpoints and CNV.A simulated model of a series of different ration of mosaism sequenced and standard curve was set up.Evaluation of 443 cases of spontaneous miscarriage samples using array-CGH and sequencing were tested for the evaluation of consistency.2.Simulated 5%,10%,15%,20%concentration of fetal cfDNA samples was set up and verified then be sequenced.3.The genomic DNA of 51 patients with mental retardation was detected by array-CGH and sequencing respectively.The detection rate and consistency of the two methods were evaluated.Result:1.Establish a data analysis process,the detection of abortion tissue evaluation results for>1Mb mutation CNV sequencing method and array-CGH detection consistent.The ROC showed that the sequencing was in good agreement with array-CGH,and the area under the curve reached 0.958.2.The simulated fetal cfDNA model was successfully establishied and 5 Mb of raw data per sample.25Mb CNV can be detected in 5%fetal concentration,7Mb CNV in 10%fetal concentration,3.5Mb CNV in 15%fetal concentration,1.8 Mb CNV in 20%fetal concentration.3.Sequencing and array-CGH were performed in detection of the cause of mental retardation.Eight cases of chromosome trisomy,1 case of haplotype and 11 cases of normal samples were detected.27 of 43 CNV fragments was pathogenic CNV,identified by sequencing with a detection rate of 100%.Conclusion:The algorithm and process of chromosomal copy number variation based on Ion Torrent high throughput sequencing platform were established.The method is expected to be used for noninvasive prenatal diagnosis and to expand the detection range of NIPT.The current data can be detected by common chromosomal microdeletions such as Cri du Chat Syndrome and angelman syndrome.It provides a new method for the detection of etiology of spontaneous abortion and mental retardation.It has the advantages of high resolution,low sample quality and low price.It can replace array-CGH as a new detection method.